PROJECT SUMMARY The innate immune system is a broad set of critical intracellular and extracellular processes that limit viral infectivity. In order to provide its essential first line of defenses against pathogens, the innate immune system must be able to accurately distinguish ?self? from foreign molecules. Misregulation of the innate immune system can cause increased persistence and susceptibility to viral infection and human diseases, such as interferonopathies. The 2?-5?-oligoadenylate synthetase (OAS) family of enzymes are important innate immune sensors of cytosolic double-stranded RNA (dsRNA). Attesting to the importance of the OAS/RNase L pathway, viruses have developed ways to evade OAS. Previous structural studies have revealed that dsRNA binding allosterically induces structural changes in OAS1 that reorganize the catalytic site to drive synthesis of 2?-5?- oligoadenylates from ATP. These 2?-5?-oligoadenylate secondary messengers activate a single known target, the latent ribonuclease (RNase L). Active RNase L in turn degrades viral and cellular RNA to halt viral replication. Although X-ray crystal structures have given some insight into how OAS1 is activated by dsRNA, we still understand very little about how specific features of the dsRNA contribute to the level of OAS1 activation. To address which specific features of dsRNA are required for potent OAS1 activation, we designed dsRNA hairpin variants, based on the RNA duplex used in the structural studies. Remarkably, while a single point mutation on one strand resulted in complete loss of OAS1 activity, the equivalent mutation on the opposite strand led to increased OAS1 activity. Despite these stark differences in ability to activate OAS1, both variants appear to bind OAS1 with similar affinity. Given these preliminary findings, I hypothesize that dsRNAs may contain competing OAS1 binding sites with remarkably different capacities to activate the protein in a context dependent manner. However, the molecular signatures defining these sites as activating and non-activating are unknown. The goal of this project is to determine how specific sequences in dsRNA, and their context, control regulation of OAS1 in the following two Specific Aims. Aim 1. To use complementary assays of OAS1 activity in vitro and in human cells to determine the features of dsRNA that lead to potent activation of OAS1. Aim 2. To use biochemical, biophysical, and structural approaches to define the molecular mechanism(s) by which the dsRNA hairpin variants differ in their effects upon OAS1 activation. These experiments will reveal new insights into the regulation of OAS1 by dsRNA. In doing so, I will enhance our understanding of host-pathogen interactions, such as how viruses might circumvent the OAS1/RNase L pathway by masking activating motifs to evade detection. My results will furthermore provide new insights into cellular translational control in the context of infection and potentially strengthen the foundations necessary to design effective treatments for viral infection.